The present invention relates to a single-chain monellin-like protein which is stable and which is at least 100-fold sweet as compared to sucrose on the weight basis. The present invention also relates to a nucleic acid encoding said monellin-like protein. Preferably, the nucleic acid further comprises a promoter and a signal sequence for directing expression and secretion of the encoded monellin-like protein in the methylotrophic yeast Pichia pastoris. The present invention further relates to a recombinant Pichia pastoris cell containing the nucleic acid encoding the monellin-like protein, a process for producing the monellin-like protein from the recombinant Pichia pastoris and product of the process.
Monellin belongs to a family of intensely sweet proteins derived from tropical plants (Dansby, Nature Biotechnology, 1997, 15:419-420). Monellin is about 3,000-fold sweet as compared to sucrose. Other similar proteins include thaumatin, miraculin, mabinlin, pentadin and aspartame (Id.) Monellin was first isolated from the West African Plant Dioscoreophyllum comminisii (U.S. Pat. Nos. 3,878,184 and 3,998,798; Morris and Cagan, Biochim. Biophys. Acta, 1972, 261:114-122). The amino acid sequence, the three-dimensional structure and various physical and chemical properties of monellin have been characterized (Ogata, et al., Nature, 1987, 328:739-742; Morris et al., J. Biol. Chem., 1973, 248:534-539; Cagan, Science, 1973, 181:32-35; Bohak and Li, Biochim. Biophys. Acta, 1976, 427:153-170; Hudson and Beeman, Biochem. Biophys. Res. Comm., 1976, 71:212-220; Van der Wel and Loeve, FEBS Lett., 1973, 29:181-183; and Frank and Zuber, HoppeSeyler""s Z Physiol. Chem., 1976, 357:585-592).
U.S. Pat. No. 4,300,576 discloses smoking articles containing thaumatin or monellin. U.S. Pat. No. 4,562,076 discloses chewing gum with coating of thaumatin or monellin. U.S. Pat. No. 4,412,984 discloses flavor potentiated oral compositions containing thaumatin or monellin. However, despite its potential as low-calorie sweeteners, wide commercial application of monellin is hampered by concerns over its poor stability to heat and pH, lack of access to sources of supply of the plant and uncertainty in the regulatory climate for food additives (Dansby, Nature Biotechnology, 1997, 15:419-420).
In 1989, Sung-Hou Kim""s group reported production of single-chain monellin in E. coli by genetic engineering (Kim et al., Protein Eng., 1989, 2:571-575). The purified single-chain monellin was found to be more heat-stable and tolerant to a wide pH range, but retained the intensity of sweetness. Several aspects of this invention have been the subject of certain U.S. patents. For example, U.S. Pat. No. 5,234,834 discloses constructs for expression of single-chain monellin in plant cells. U.S. Pat. No. 5,487,923 discloses a sweet proteinaceous compound of the formula B-C-A, wherein B represents a peptide portion at least 90% homologous to residues 1-46 of the B chain of native monellin and modified only by conservative substitutions; C is a covalent bond or is a hydrophilic, physiologically acceptable covalent linker capable of providing a spacing length equivalent to a peptide of 1-10 amino acids selected so as to reside on the external portion of the molecule and not to disturb the native conformation; and A represents a peptide at least 90% homologous to residues 6-45 of the A chain of native monellin and modified only by conservative substitution. U.S. Pat. No. 5,487,983 discloses an expression system for making the single-chain monellin disclosed in U.S. Pat. No. 5,487,923. U.S. Pat. No. 5,670,339 discloses DNA encoding the single-chain monellin disclosed in U.S. Pat. No. 5,487,923. U.S. Pat. No. 5,672,372 discloses methods for sweetening a food composition with the single-chain monellin disclosed in U.S. Pat. No. 5,487,923. U.S. Pat. No. 5,264,558 discloses a single-chain monellin protein that is, in a standard taste test, at least 50 times that of sucrose on a weight basis.
Recently, Kondo et al., Nature Biotechnology, 1997, 15:453-457 discloses heterologous expression of a single-chain monellin protein in the yeast Candida utilis intracellularly. It reports that monellin was produced at a high level, accounting for  greater than 50% of the soluble protein.
The methylotrophic yeast Pichia pastoris has been used as a protein expression system. Several aspects of this expression system have been the subject of certain U.S. patents. For example, U.S. Pat. No. 4,837,148 discloses autonomous replication sequences for Pichia pastoris. U.S. Pat. No. 4,855,231 discloses regulatory region for heterologous gene expression in Pichia pastoris cells. U.S. Pat. No. 4,882,279 discloses site selective genomic modification of Pichia pastoris. U.S. Pat. No. 4,929,555 discloses a method for making whole cells of Pichia pastoris competent for transformation. U.S. Pat. No. 5,122,465 discloses a process for generating a selectable phenotype in strains of Pichia pastoris. U.S. Pat. No. 5,324,639 discloses production of insulin-like growth factor-1 in methylotrophic cells, including Pichia pastoris cells.
A number of signal sequences have been used to direct secretion of heterologous proteins expressed in Pichia pastoris cells. Examples of such signal sequences include, but are not limited to, the signal sequence of Pichia pastoris acid phosphatase, the signal sequence of Aspergillus giganteus alpha-Sarcin (Martinez-Ruiz et al., Protein Expr. Purif., 1998, 12(3):315-22; Abdulaev et al., Protein Expr. Purif., 1997, 10(1):61-9; Kotake et al., J. Lipid Res., 1996, 37(3):599-605), the signal sequence of alpha-N-Acetylgalactosaminidase (alphaNAGAL, EC 3.2.1.49) (Zhu et al., Arch. Biochem, Biophys., 1998, 352(1):1-8), the signal peptide of the OmpA protein (Heim et al., Biochim. Biophys. Acta., 1998, 1396(3):306-19), the signal sequence of the mouse alpha-factor signal (cCell) or the native signal sequence of pepper endo-beta-1,4-glucanases (Ferrarese et al., FEBS Lett., 1998, 422(1):23-6), signal peptide of laccase isolated from the ligninolytic fungus Trametes (Jonsson et al., Curr. Genet., 1997, 32(6):425-30), signal peptide of murine lysosomal acid alpha-mannosidase (Merkle et al., Biochim. Biophys. Acta., 1997, 1336(2):132-46), signal peptide of the porcine inhibitor of carbonic anhydrase (Wuebbens et al., Biochemistry, 1997, 36(14):4327-36), signal sequence of Aspergillus awamori glucoamylase (Fierobe et al., Protein Expr. Purif., 1997, 9(2):159-70), signal sequence of mouse major urinary protein (Ferrari et al., FEBS Lett., 1997, 401(1):73-7), signal sequence of pho1 (Skory et al., Curr. Genet., 1996, 30(5):417-22), signal sequence of rabbit angiotensin-converting enzyme (ACE) (Sadhukhan et al., J. Biol. Chem., 1996, 271(31):18310-3), prepeptide sequence of Pichia pastoris aspartic proteinase (Tsujikawa et al., Yeast, 1996, 12(6):541-53), signal sequence of Pichia pastoris PRC1 (Ohi et al., Yeast, 1996, 12(1):31-40), the signal sequence of a bacterial thermostable alpha amylase and SUC2 gene signal sequence from Saccharomyces cerevisiae (Paifer et al., Yeast, 1994, 10(11):1415-9) and the signal sequence of Saccharomyces cerevisiae mating pheromone xcex1-factor (Fidler et al., J. Mol. Endocrinol., 1998, 21(3):327-336).
Although the methylotrophic yeast Pichia pastoris has been used successfully for the production of various heterologous proteins, U.S. Pat. No. 5,324,639 discloses that at the present level of understanding of methylotrophic yeast expression systems, it is unpredictable whether a given gene can be expressed to an appreciable level in such yeast or whether the yeast host will tolerate the presence of the recombinant gene product in its cells. U.S. Pat. No. 5,324,639 further discloses that it is especially difficult to foresee if a particular protein will be secreted by the methylotrophic yeast host, and if it is, at what efficiency. For example, Vollmer et al., J. Immunol. Methods, 1996, 199(1):47-54, reports that when the 323 amino acid residues of the human sIL-6R are inserted into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris, no detectable expression and secretion of the recombinant protein was obtained. Up to date, monellin has not been expressed and secreted using the Pichia pastoris expression system.
Given the great interest in the commercial application of monellin, there is a great need for a more efficient method for producing stable monellin which still retains its native sweet intensity and which simplify down stream purification procedures. The present invention addresses these and other needs in the art. Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein, said chimeric protein comprises, from N-terminus to C-terminus: a) a first peptidyl fragment consisting of an amino acid sequence that has at least 40% identity to residues 1-50 of the B chain of native monellin, in which the percentage identity is determined over an amino acid sequence of identical size to the B chain of native monellin; b) a peptidyl bond, or a second peptidyl fragment consisting of 1-12 amino acids; and c) a third peptidyl fragment consisting of an amino acid sequence that has at least 40% identity to residues 1-45 of the A chain of native monellin, in which the percentage identity is determined over an amino acid sequence of identical size to the A chain of native monellin, wherein said chimeric protein is stable and a given amount of said chimeric protein is at least 100-fold sweet as compared to the identical amount of sucrose, and within said nucleic acid, codons which are preferably used by yeast cells are used. Preferably, the isolated nucleic acid further encodes a promoter which is capable of directing protein expression in Pichia pastoris and/or an amino acid sequence which is capable of directing secretion of the encoded chimeric protein from Pichia pastoris. 
The present invention also relates to a recombinant Pichia pastoris cell containing the above nucleic acids. The present invention further relates to a process for producing a chimeric protein comprising growing a recombinant Pichia pastoris cell containing the above nucleic acid such that the encoded chimeric protein is expressed and secreted by the cell, and recovering the expressed and secreted chimeric protein. Finally, the present invention relates to products of the above processes.